The Laboratory Zebrafish - LABSG Laboratory Zebrafish Workshop in Lab Animal Medicine May 19, 2011 David M. Kurtz, DVM, PhD Disclaimers • This is not an ACLAM sanctioned presentation

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The Laboratory Zebrafish

Workshop in Lab Animal Medicine

May 19, 2011

David M. Kurtz, DVM, PhD

Disclaimers

• This is not an ACLAM sanctioned presentation

• All information is deemed reliable and correct• No warranty for accuracy

• No information presented is known to be specifically included in ACLAM Board examinations

Acknowledgments

• Julia Whitaker - UNC

• Diane Forsythe & Mary Grant – NIEHS

• Mary Ann Vasbinder - GSK


Genus & Species?


www.azeem-photos.blogspot.com


www.azeem-photos.blogspot.com

Kingdom Animalia

Phylum Chordata

Order Cypriniformes

Family Cyprinidae

Genus Danio

Species rerio

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Guidance on the housing and care of Zebrafish - Danio rerio

Zebrafish Characteristics

• Freshwater – Ganges River (India)

• Teleost (vertebrate)

• Sexually dimorphic (not always easy)• Females larger, more silver, rounded

• Males- more brightly colored, streamlined

http://dir.nichd.nih.gov/lmg/uvo/WEINSLAB.html

♀

♂

Zebrafish Characteristics

• Adults 3-4 cm long

• Lifespan (laboratory): ~ 3.5 years

• Sexual maturity: 6-8 months of age

• Optimal breeding: 6 – 18 months

• Like most fish – possess a lateral line – series of mechanoreceptors used to sense environmental conditions

• Lack teeth – instead have pharyngeal jaw associated with posterior gill arch used to grind food

Nutrition

• Omnivorous

• Natural diet – zooplankton, insects, algae, invertebrate eggs

• Begin feeding 5 days post fertilization (dpf)• Yolk sac – depleted around 7-8 dpf

• Mouth opens

• Swim bladder inflates

• Development of digestive tract completeYolk sac

Nutrition

• Laboratory diet• Larvae 5-14 dpf – Paramecia or rotifers

• > 14 dpf to adult – flake or pellets

• Do not feed more than can be consumed in 3-5 minutes. Excess = nitrogenous waste

• Artemia – Brine shrimp• Start at 28 dpf

• Optimal for breeding colonies

Aquatic Habitats

Housing

• Holding tanks• Static – aquarium – OK for small populations

• Flow through – lowest levels of nitrogenous waste but uses a lot of water

• Recirculating – most common

• Housing density ~ 5 adults/liter

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Housing

Recirculating Systems

Water Quality

Most important aspect of research aquaculture systems

Water Source

• Municipal water – chemically treated to remove chlorine, chloramines, copper

• Sodium thiosulfate• Activated charcoal

• Wells• few infectious agents or chemicals• Small volumes

• Ponds• not recommended• Lots of pathogens• Chemical contamination from runoff

• Distilled• Must add back salts• expensive for large volumes

• Reverse osmosis filtration• Must add back salts• most common in research aquaculture

Water Quality

• What is the name of the system component indicated by the red arrow?

• What process primarily takes place here?

Water Quality

• Biological Filter

• Nitrogen of Nitrification Cycle

Nitrogen Cycle

Ammonia (NH3)

Toxic

Nitrite (NO2)

Toxic

Nitrate (NO3)

Non-ToxicNitrosomonas spp. Nitrobacter spp.

Excretory Waste Excess Feed

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Water Quality

• Biological Filter

• Nitrogen of Nitrification Cycle

Other types of filtration?• Mechanical

• Chemical

Environmental Conditions

• Temperature• 210 – 320C (700 – 900F)• 280C (830F) – optimal• Limit rapid changes ± 1.50C/day

• pH• Range of 7-8 – preferred 7.0• ph > 8.0 favors NH4

+ → NH3 – more toxic• pH < 5.0 inhibits nitrifying bacteria – ammonia

accumulation

Environmental Conditions

• Salinity ≈ Conductivity – total dissolved solids (ions)• Hardness [Ca++ or Mg++ ] – 80-200 ppm• Important when using distilled or RO source water

• Dissolved Oxygen (DO) – 6.0 ppm• Too low – hypoxia, fish gather at surface• Too high?

• Light cycles – 14 light: 10 dark most common• Gradual brightening and dimming over 30 minutes

recommended

Zebrafish Reproduction

• Oviparous – egg layers• Broadcast spawners• Females produce 100-200 eggs every 1-3 days• Females prefer to release eggs over a substrate

(e.g., plants, rocks, marbles)• Adults will eat eggs if easily accessible

http://www.tecniplast.it/aquatic/zebtec.php

http://www.aquatichabitats.com/breeder_tank_small.htm


Egg Collection


• Large Scale egg production

• Holds up to 2000 breeding adults

MEPS - Aquatic Habitats

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Research Uses

• Vertebrate embryonic development

• Gene function analysis

• Mutagenesis

• Toxicology

Zebrafish Models

• Small size, little maintenance • 1/1000th the cost of mice (debatable)

• Synchronized embryonic development

• External development- view all stages of development (transparent eggs)

• Rapidly develop - hatch in ~ 72 hours

• Genome half the size of mammals’

• Genome sequenced

Embryonic development

• 45 min- first cleavage

• Gastrulation (5.5 – 10 hpf)• Endoderm, mesoderm, ectoderm

• 24 hpf- heart beat, blood circulation, responsive to touch

http://zfin.org/zf_info/zfbook/stages/index.html

Embryonic development

Normal zebrafish heart beat

http://www.exploratorium.edu/imaging_station/gallery.php?Asset=Normal%20zebrafish%20heartbeat&Group=&Category=Zebrafish&Section=Introduction

Zebra embryo development

Movie by Tyler and Kozlowski- 2.64 MB

http://www.devbio.uga.edu/movies/files/Fish/TylerKozlowski%20fish2.mov

Genetic Models

• Over 4000 reported genetically modified lines

• Spontaneous mutations

• Mutagenized models• ENU

• Radiation

• Transgenics

See-through Zebrafish

Richard White, MD, PhD

Watch heartbeat:http://www.childrenshospital.org/research/Site2029/mainpageS2029P22sublevel57.html

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Transgenic Zebrafish

http://ame2.asu.edu/sites/eighthday/documentation/gfpecology/ http://stemcells.ucsd.edu/imagegallery.asp

Credit: David Traver and Ken Poss Credit: Hao Zhu and Leonard Zon

• Transgenic Reporter lines• Green Fluorescent Protein (GFP)• Red Fluorescent Protein (RFP)

Clinical Exam

• Water Quality • Present and historical – importance of good

records

• Behavior• Swimming, respiration, clustering

• Clinical signs• Opercular flaring• Petechia or hemorrhage• Change in body color• Scale loss• Improper buoyancy• Lethargy/decreased swimming• Surface breathing

Clinical Exam

• Skin/mucus scrape

• Gill biopsy

• Fin clip

• GI wet mount (fecal)

• Blood sample

Pictures from “Clinical Diagnostic Techniques in Fish” by Carol Poll, C L Davis Lab Animal seminar in Chicago, April 2005

Zebrafish Necropsy

Zebrafish Necropsy

Zebrafish Necropsy

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Zebrafish Necropsy

Zebrafish Necropsy

Diseases

• Water Quality – chemical toxicity

• Infectious disease

Diseases Associated with Water Quality

• Chlorine

• Copper

• Ammonia

• Nitrite

• Dissolved Gases

Chlorine Toxicity

• Municipal water contains 0.5-1.0 ppm

• Failure of water purification system

• All fish are very sensitive

• Acute gill necrosis • Opercular flaring

• Hypoxia

• Cherry red gills

• Petechial hemorrhage around head

Copper Toxicity

• Leaching from copper piping (especially new systems)

• Gill damage

• Affects reproductive development

• Increased susceptibility to infectious disease

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Ammonia Toxicity

• Waste product (fish and feed)• Highly toxic• Elevations usually due to abrupt change to

system especially affecting biological filter (biofilter “crash”)

• Effects observed at levels 0.002 ppm. Should avoid > 1.0 ppm

• Primary effects on CNS (seizures)• Hyperexcitable, anorexia, reduced growth• Histo – gill hyperlasia (chronic exposure)

Nitrite Toxicity

• Usually in conjunction with Ammonia toxicity

• Another name for nitrite toxicity in fish?

Nitrite Toxicity

• “Brown blood disease” – Methemoglobinemia

• Reported in catfish – rare in research setting

• Nitrite crosses the gills and oxidizes hemoglobin• Hypoxia

Name the Disease

http://zebrafish.org/zirc/health/Disease_imageshttp://oregonstate.edu/dept/salmon/projects/salmon-idiopathic.html

http://www.lrf.org/TMDL2print/TMDL03print.gif

Gas Bubble Disease (GBD)

• Supersaturation of dissolved gases (02 or N2)

• “Bends” in people

• Gas forms bubbles in tissues resulting in damage

• May not be apparent grossly – requires histology• May dissipate within 24 hours post-mortem

• Most often caused by leak in recirculating system resulting in air sucked into supply by high pressure pumps

Skinny Zebrafish with curved spine

Most likely etiologic diagnosis?

Zebrafish International Resource Center (ZIRC), University of Oregon

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Skinny Zebrafish with curved spine

Pseudoloma neurophilia


Pseudoloma neurophilia

• Neuronal microsporidia• Obligate intracellular parasite• High prevalence in research colonies

(>50% tested by ZIRC) • Severe emaciation• Curvature of spine in older adults• Reduced growth and fecundity• Can cause high mortality in fry• Possible vertical transmission

• Cannot be eliminated via bleaching eggs

• No treatment – test (PCR) and cull

H&E

Acid FastXenomas


Mycobacteriosis

• What are the 3 most common species of atypical Mycobacterium in Zebrafish?

M. Kent, University of Oregon

Mycobacteriosis

• Mycobacterium marinum

• M. fortuitum

• M. chelonae (chelonei)

Mycobacteriosis

• Rod-shaped bacteria• Multi-organ granulomas - Liver, spleen, kidney,

repro organs• High prevalence in research colonies (> 30%

tested by ZIRC)• Long incubation, chronic, subclinical• Clinical signs:

• Wasting, emaciation• Acutely: “dropsy”• Petechia• Ulceration of scales, fin erosion

http://edis.ifas.ufl.edu/VM055http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1695495

Mycobacteriosis

Granulomas

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Dropsy

http://www.myoops.org/twocw/tufts/courses/5/content/D215706/C43031.jpg

http://www.ucs.louisiana.edu/~jlm2431/dropsy.jpg

• General term for abdominal distension and scale edema

• Usually due to kidney or gill failure and loss of osmoregulation and hypoproteinemia

Mycobacteriosis

Special stain for Mycobacterium?

Mycobacteriosis

• Acid-fast

• Ex. Fite-Faraco, Ziehl-Neelsen

Mycobacteriosis- acid fast

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1695491 Dr. Virginia Godfrey, UNC

Mycobacteriosis

• No effective treatment

• Eradication of infected stocks

• Disinfect tank and filter system • With bleach for 3 days, rinse for 3 days

• Restock with fish, test for reinfection

Mycobacteriosis- Zoonotic

• Cutaneous infections in humans

• “Fish handler’s granuloma” or “swimmer’s granuloma”

• Usually local and self-limiting, life-threatening if immunocompromised

• Wear gloves!!

http://www.aquatouch.com/images/Granuloma.jpg

http://www.reefkeeping.com/issues/2003-07/sp/feature/index.php

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Pseudocapillaria tormentosa

http://zebrafish.org/zirc/health/Disease_images/CapillariaWetMounts4.jpg

• Intestinal Nematode• Common in research colonies

(>24% of facilities tested by ZIRC)• Most infections are subclinical• Wasting and emaciation• Associated with penetrating GI

infection and neoplasia• Dx: GI wet mount - eggs or adult • Tx: trichlorfonmebendazole,

Panacur, ivermectin

What is the anesthetic/euthanasia agent most commonly used in Zebrafish?

Anesthesia/Euthanasia


• MS- 222 (tricaine methanesulfonate)• 0.05 - 0.15 mg/ml - anesthesia

• 2.0 - 2.5 mg/ml – euthanasia

• 10-20 minute emersion for euthanasia

• Must buffer with sodium bicarbonate b/c low pH

Pictures from “Clinical Diagnostic Techniques in Fish” by Carol Poll, C L Davis Lab Animal seminar in Chicago, April 2005


• Benzocaine hydrochloride• 25-100 mg/L for anesthesia• Often used in field studies• Small margin of safety

• Clove Oil• Eugenol or Isoeugenol (90-95%)• Reduced cost• Often used in field studies• Not FDA approved for use in fish

Euthanasia

• Rapid Cooling• Currently not recommended in 2007 AVMA Guidelines on

Euthanasia• Recent publications advocate euthanasia via rapid cooling

for Zebrafish (warm water species only) over MS-222• Wilson JM et al., Comp Med 48(6), November 2009• Blessing JJ at al., J Fish Biol 76, 2010

• Current NIH Guidelines • Submersion in ice water (5 parts ice/1 part water; 0-40C) for at least

10 minutes (20 minutes if unable to visualize opercular movement and for fry 4-7 dpf)

• Deviation from Guide standards?? – IACUC approval

Regulatory and IACUC Issues

• Specific aquatic animal section in 2010 Guide (CH 3 - Environment, Housing and Management)

• Field studies

• Animal tracking & records

• Facility inspections – especially outside main vivarium

• Euthanasia

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Zebrafish International Resource Center (ZIRC) 5274 University of OregonEugene, OR 97403-5274, USAPhone: 541-346-6028Email: [email protected]: http://zebrafish.org

http://www.zhaonline.org/

Zebrafish References

http://www.zhaonline.org/Guidance_on_the_housing_and_care_of_zebrafish__2010_.pdf

Questions

www.exploratorium.edu

www.telegraph.co.uk

Additional Fish Disease Slides

From J. Whitaker talk 2008and

ZIRC

Velvet Disease

• Piscinoodinium pillulare• Parasitic dinoflagellate• Infects the skin and gills – may

see grey or rust colored sheen• Clinically - lethargy,labored

breathing, stays near water surface

• Wet mount of skin scraping or fin clip

• Histological sections

http://www.petfish.net/articles/pix/arts3/velvet.jpg

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Piscinoodinium pillulare

http://www.reefs.org/library/aquarium_net/0597/images/img0077.jpg

http://fishweb.ifas.ufl.edu/AquaticAnimalHealth/ExamplesFishDiseases.htm

Pleistophora hyphessobryconis

• Microsporidia

• Neon Tetra disease

• Seen in multiples aquarium species – and some research institutions

• Primarily affect skeletal muscle

• Diagnosis only via histology (no PCR yet)

Zebrafish International Resource Center

Egg Associated Inflammation and Fibroplasia

• Females with extended abdomens

• Common – especially aging females

• Cause unknown – egg retention and degeneration

• Grossly – enlarged tumor-like ovaries often with adhesions

• Histo – severe inflammation (arrows) +/- fibrosis

Mike Kent, University of Oregon

Zebrafish International Resource Center

Bacterial Septicemia

• Streptococcus iniae

• Edwardsiella tarda

• Aeromonas hydrophila

• Pseudomonas spp.

• Flavobacterium columnare– Previously Flexibacter columnaris

• Flavobacterium spp.

• Which are zoonotic?

Bacterial Septicemia

• Streptococcus iniae (zoonotic)

• Edwardsiella tarda (zoonotic)

• Aeromonas hydrophila (zoonotic)

• Pseudomonas spp. (zoonotic)

• Flavobacterium columnare– Previously Flexibacter columnaris

• Flavobacterium spp.

Clinical signs

• Exophthalmos

• increased respiratory effort

• Dropsy/abdominal distension

• Petechial hemorrhages‐ on body, around eyes, mouth, anus, opercula, or fin base

• Necropsy: Congested, hemorrhagic organs, pale liver, dark red spleen, blood‐tinged peritoneal fluid

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Diagnosis & treatment

• Isolation in broth or on blood agar

– Grow at 20‐ 25°C 24 – 48 hr

• Antibiotics‐ usually bath immersion

– Sulfadiazine‐trimethoprin

– Enrofloxacin

– Oxytetracycline

Flavobacterium columnare

http://zebrafish.org/zirc/health/Disease_images/TailRot4.jpg

http://www.klamathwaterquality.com/fish_disease_close_up_gill_shot.jpg


http://www.umesc.usgs.gov/aquatic/drug_research/disease_model/images/haystacks_columnaris.jpg

http://www.vet.uga.edu/VPP/Undergrad/Siegel/index.php


http://www.vet.uga.edu/VPP/Undergrad/Siegel/index.php

Aquatic Mycosis

• Saprolegnia spp.

• Oomycetes

• Opportunistic‐ damaged skin, poor husbandry, concurrent infection

• Signs: thin, white filaments building up to white “cottony” mattes

Saprolegnia

• Dx: skin wet mounts

• Broad aseptate hyphae ± sporangia on tips

• Hyphae visible on H & E

• Pronounced with silver stain

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Saprolegnia

• Ubiquitous

• Spread rapidly

• Debilitation: secondary bacterial sepsis & disrupted osmotic balance

• Tx: saltwater bath, formalin bath

• Best Tx‐malachite green‐ (but teratogen & mutagen)

Saprolegnia

http://www.americanaquariumproducts.com/images/graphics/saprolegniafungus.jpg

http://www.aquarium.lt/files/articles/ligos/saprolegnia_i.jpg

http://www.ncwildlife.org/pg03_Fishing/images/img_3g1_sapro.jpg

http://www.uib.no/bot/kurs/BB220/Algae/Figures/fish.jpg

Saprolegnia

http://kentsimmons.uwinnipeg.ca/16cm05/16labman05/lb2pg3_files/saprolegniazoosporangium.gif

http://www.fishlife.nl/saprolegnia%202.jpg

White Spot Disease

• A.k.a.?

• Caused by?

White Spot Disease

• Ich

• Ichthyophthirius multifilis

White Spot Disease

• Ciliated protozoan

• Trophont colonizes epithelium

– Fins, skin, gills‐ feeds on tissue & fluids

• Trophont ruptures overlying epithelium → attaches to substrate

• Develop into free‐swimming theronts‐must colonize host within 48 hrs

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Ich

• Multifocal, raised, white mucoid nodules on skin and gills

• Flashing & increased respiratory effort

• Dx: skin wet mounts

– Trophont with horseshoe‐shaped nucleus

• Tx: Raise temp 1.5°C every 24‐36 hr for 3 days to speed up life cycle plus salt bath, formalin bath

Ich

http://www.fishpalace.org/Disease.html#Septicemia

http://www.vet.cornell.edu/PUBLIC/FishDisease/AquaticProg/highlights/Ich/Ich.htm

http://www.guppyendler.com/upload/enfermedades/Ichthyophthirius_multifiliis_1.jpg

Ich

http://fishweb.ifas.ufl.edu/AquaticAnimalHealth/AnimalHealthPix/Ichthyophthirius.jpg

http://www.reefs.org/library/aquarium_net/0197/images/img0006.jpg

http://www.cascadekoi.com/images/body/Koi/parasites/WhiteSpot%20Image.jpg

http://www.fish-helpline.co.uk/photos/Micro03.jpg

http://www.koidream.com/ZiektenWstip4.jpg

Trichodinosis

• Trichodina spp. (+ 5 other genera)

• Ciliated protozoan

• Skin‐associated‐ broad host range, gill‐associated‐ more host‐specific

• Direct life cycle

• Don’t persist in environment (>48 hr)

Trichodina

• Heavy infection: poor body condition, low reproductive rates, mortality

• Signs: respiratory distress, skin erosion, excess mucus, sloughed scales, frayed fins

• Histo: hyperplastic gill epithelium

Trichodina

• Dx: wet mount‐ skin scrape or gill clip

• Circular, dome‐shaped

• Concentric rows of cilia ± dentricles

• Rotating motion

• Usually secondary: poor water quality, high stocking density, concurrent infection, gill damage

• Tx: salt bath, formalin

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Trichodina

http://www.cnr.vt.edu/fisheries/afs/fisheries_techniques/Chapter14/Skin%20wet%20mount%20of%20catfish%20with%20trichodina.jpg

http://home.infostations.net/letitiashen/Fish/trichodina3.jpg

“Clinical Diagnostic Techniques in Fish” by Carol Poll, C L Davis Lab Animal seminar in Chicago, April 2005

Viruses

• Infectious Pancreatic Necrosis Virus

– Salmonids (trout & salmon)

– Birnavirus‐ ds RNA

– Subclinical in zebrafish (us. wild‐type)

• Iridoviruses (goldfish)

• Rhabdoviruses (carp, cichlids)

Helpful links• http://zfin.org/cgi‐bin/webdriver?MIval=aa‐ZDB_home.apg

• http://zebrafish.org/zirc/health/diseaseManual.php

• Fish disease presentation:

– http://wfs.sdstate.edu/wfsdept/courses/WL423_523/DISEASE2.PPT

• http://www.exploratorium.edu/

• Goldfish & koi diseases:

– http://www.mu.edu/~buxtoni/puregold/disease/symptom/byname.htm

• http://www.aquaworldnet.com/awmag/diseases.htm

• Great pictures: http://www.dpi.nsw.gov.au/__data/assets/pdf_file/0005/209309/Silver‐Perch‐Diseases‐Manual.pdf

• Video of necropsy:

– http://aquanic.org/real/necropsy/

Useful References

• Fish Medicine by Stoskopf

• Fish Disease (Diagnosis and Treatment) by Edward Noga

• March 2002 Lab Animal journal (fish)

• ALN Magazine vol 7, No 2 March 2008‐Aquatic Facilities

Documents

The Laboratory Zebrafish - LABSG Laboratory Zebrafish Workshop in Lab Animal Medicine May 19, 2011 David M. Kurtz, DVM, PhD Disclaimers • This is not an ACLAM sanctioned presentation